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ARTICLE
Year : 2016  |  Volume : 5  |  Issue : 5  |  Page : 138-139

Effects of sorbitol and glycerol on the structure, dynamics, and stability of Mycobacterium tuberculosis pyrazinamidase


1 Department of Biology, Faculty of Science, Azarbaijan Shahid Madani University, Tabriz, Iran
2 Department of Life Science Engineering, Faculty of New Sciences & Technologies, University of Tehran, Tehran, Iran
3 Department of Microbiology, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Date of Web Publication17-Feb-2017

Correspondence Address:
Farahnoosh Doustdar
Department of Microbiology, Faculty of medicine, Shahid Beheshti University of Medical sciences, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.1016/j.ijmyco.2016.10.024

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  Abstract 


Objective/background: Mycobacterium tuberculosis pyrazinamidase (PZase) is known an enzyme that is involved in degradation of pyrazinamide to ammonia and pyrazinoic acid. Pyrazinamide is an important first-line drug used in the short-course treatment of tuberculosis. Previous investigations have indicated that the pyrazinamide (PZA)-resistant M. tuberculosis strains are caused by point mutations in the PZase enzyme which is the activator of the prodrug PZA. Although the general fold of PZase was determined, the structural and functional properties of the enzyme in solution were not understood very well. In this study, the PZase enzyme was overexpressed and purified. In addition, two polyols, namely sorbitol and glycerol, were chosen to study their effects on the structure, dynamics, and stability of the enzyme. To gain a deeper insight, molecular dynamics simulation and spectroscopic methods, such as fluorescence spectroscopy and circular dichroism (CD), were used.
Methods: The genes were cloned in Escherichia coli BL21 (DE3), harboring the recombinant pET-28a (+) plasmid, overexpressed and purified by Ni-NTA Sepharose. The far UV–visible CD spectra were measured by a Jasco-810 spectropolarimeter. The intrinsic fluorescence spectra were measured on a Cary Varian Eclipse spectrofluorometer. For molecular dynamics (MD) simulations, we have applied GROMACS4.6.5.
Results: The results showed that glycerol and sorbitol increased the enzyme activity up to 130% and 110%, respectively, at 37°C. The stability of PZase was decreased and the half-life was 20 min. Glycerol and sorbitol increased the PZase half-life to 99 min and 23 min, respectively. The far UV CD measurements of PZase indicated that the CD spectra in glycerol and sorbitol give rise to an increase in the content of α-helix and β-sheets elements. The average enzyme root mean square deviation (RMSD) in sorbitol solution was about 0.416nm, a value that is higher than the enzyme RMSD in the pure water (0.316). In dictionary of protein secondary structure (DSSP) results, we observed that the secondary structures of the protein are partially increased as compared to the native state in water. The experimental and simulation data clearly indicated that the polyols increased the PZase stabilization in the order: glycerol>sorbitol.
Conclusion: It can be concluded that the native conformation of the enzyme was stabilized in the sorbitol and glycerol and tend to exclude from the PZase surface, forcing the enzyme to keep it in the compactly folded conformation. The glycerol molecules stabilized PZase by decreasing the loops flexibility and then compacting the enzyme structure. It appears that more stability of PZase in glycerol solution correlates with its amphiphilic orientation, which decreases the unfavorable interactions of hydrophobic regions.

Keywords: Protein stability, Pyrazinamidase, Simulation, Tuberculosis


How to cite this article:
Khajehzadeh M, Mehrnejad F, Pazhang M, Doustdar F. Effects of sorbitol and glycerol on the structure, dynamics, and stability of Mycobacterium tuberculosis pyrazinamidase. Int J Mycobacteriol 2016;5, Suppl S1:138-9

How to cite this URL:
Khajehzadeh M, Mehrnejad F, Pazhang M, Doustdar F. Effects of sorbitol and glycerol on the structure, dynamics, and stability of Mycobacterium tuberculosis pyrazinamidase. Int J Mycobacteriol [serial online] 2016 [cited 2020 Aug 7];5, Suppl S1:138-9. Available from: http://www.ijmyco.org/text.asp?2016/5/5/138/200381




  Conflict of interest Top


None declared.





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